Nearly monodisperse Au nanorods (NRs) with different aspect ratios were separated from home-synthesized polydisperse samples using a gradient centrifugation method. The morphology, size and its distribution, and photo-absorption property were analyzed by transmission electron microscopy, atomic force microscopy and UV-visible spectroscopy. Subsequently, using colloidal Au NRs (36.2 nm ×10.7 nm) with 97.4% yield after centrifugation and Au nanospheres (NSs) (22.9 ± 1.0 nm in diameter) with 97.6% yield as Au substrates, surface-enhanced Raman scattering (SERS) spectra of 2,4-dichlorophenoxyacetic acid (2,4-D) were recorded using laser excitation at 632.8 nm. Results show that surface enhancement factors (EF) for Au NRs and NSs are 6.2 × 10(5) and 5.7 × 10(4) using 1.0 × 10(-6) M 2,4-D, respectively, illustrating that EF value is a factor of ∼10 greater for Au NRs substrates than for Au NSs substrates. As a result, large EF are a mainly result of chemical enhancement mechanisms. Thus, it is expected that Au NPs can find a comprehensive SERS application in the trace detection of pesticide residues.